Proteasome Inhibitors

ABSTRACT

The present invention provides novel compounds useful as proteasome inhibitors. The invention also provides pharmaceutical compositions comprising the compounds of the invention and methods of using the compositions in the treatment of various diseases.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/964,708, filed Aug. 12, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/209,511, filed Aug. 15, 2011, now U.S. Pat. No.8,530,694, which is a continuation of U.S. patent application Ser. No.12/704,830, filed on Feb. 12, 2010, now U.S. Pat. No. 8,003,819, whichis a continuation of U.S. patent application Ser. No. 12/217,243, filedon Jul. 2, 2008, now U.S. Pat. No. 7,687,662, which is a continuation ofU.S. patent application Ser. No. 11/890,412, filed on Aug. 6, 2007, nowU.S. Pat. No. 7,442,830 B1, of which each application is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to boronic acid and boronic estercompounds useful as proteasome inhibitors. The invention also providespharmaceutical compositions comprising the compounds of the inventionand methods of using the compositions in the treatment of variousdiseases.

BACKGROUND OF THE INVENTION

Boronic acid and ester compounds display a variety of pharmaceuticallyuseful biological activities. Shenvi et al., U.S. Pat. No. 4,499,082(1985), discloses that peptide boronic acids are inhibitors of certainproteolytic enzymes. Kettner and Shenvi, U.S. Pat. No. 5,187,157 (1993),U.S. Pat. No. 5,242,904 (1993), and U.S. Pat. No. 5,250,720 (1993),describe a class of peptide boronic acids that inhibit trypsin-likeproteases. Kleeman et al., U.S. Pat. No. 5,169,841 (1992), disclosesN-terminally modified peptide boronic acids that inhibit the action ofrenin. Kinder et al., U.S. Pat. No. 5,106,948 (1992), discloses thatcertain boronic acid compounds inhibit the growth of cancer cells.Bachovchin et al., WO 07/000,5991, discloses peptide boronic acidcompounds that inhibit fibroblast activating protein.

Boronic acid and ester compounds hold particular promise as inhibitorsof the proteasome, a multicatalytic protease responsible for themajority of intracellular protein turnover. Adams et al., U.S. Pat. No.5,780,454 (1998), describes peptide boronic ester and acid compoundsuseful as proteasome inhibitors. The reference also describes the use ofboronic ester and acid compounds to reduce the rate of muscle proteindegradation, to reduce the activity of NF-κB in a cell, to reduce therate of degradation of p53 protein in a cell, to inhibit cyclindegradation in a cell, to inhibit the growth of a cancer cell, and toinhibit NF-κB dependent cell adhesion. Furet et al., WO 02/096933,Chatterjee et al., WO 05/016859, and Bernadini et al, WO 05/021558 andWO 06/08660, disclose additional boronic ester and acid compounds thatare reported to have proteasome inhibitory activity.

Ciechanover, Cell, 79: 13-21 (1994), discloses that the proteasome isthe proteolytic component of the ubiquitin-proteasome pathway, in whichproteins are targeted for degradation by conjugation to multiplemolecules of ubiquitin. Ciechanover also discloses that theubiquitin-proteasome pathway plays a key role in a variety of importantphysiological processes. Rivett et al., Biochem. J. 291:1 (1993)discloses that the proteasome displays tryptic-, chymotryptic-, andpeptidylglutamyl peptidase activities. Constituting the catalytic coreof the 26S proteasome is the 20S proteasome. McCormack et al.,Biochemistry 37:7792 (1998), teaches that a variety of peptidesubstrates, including Suc-Leu-Leu-Val-Tyr-AMC, Z-Leu-Leu-Arg-AMC, andZ-Leu-Leu-Glu-2NA, wherein Suc is N-succinyl, AMC is7-amino-4-methylcoumarin, and 2NA is 2-naphthylamine, are cleaved by the20S proteasome.

Proteasome inhibition represents an important new strategy in cancertreatment. King et al., Science 274:1652-1659 (1996), describes anessential role for the ubiquitin-proteasome pathway in regulating cellcycle, neoplastic growth and metastasis. The authors teach that a numberof key regulatory proteins, including, cyclins, and the cyclin-dependentkinases p21 and p27^(KIP1), are temporally degraded during the cellcycle by the ubiquitin-proteasome pathway. The ordered degradation ofthese proteins is required for the cell to progress through the cellcycle and to undergo mitosis.

Furthermore, the ubiquitin-proteasome pathway is required fortranscriptional regulation. Palombella et al., Cell, 78:773 (1994),teaches that the activation of the transcription factor NF-κB isregulated by proteasome-mediated degradation of the inhibitor proteinIκB. In turn, NF-κB plays a central role in the regulation of genesinvolved in the immune and inflammatory responses. Read et al., Immunity2:493-506 (1995), teaches that the ubiquitin-proteasome pathway isrequired for expression of cell adhesion molecules, such as E-selectin,ICAM-1, and VCAM-1. Zetter, Seminars in Cancer Biology 4:219-229 (1993),teaches that cell adhesion molecules are involved in tumor metastasisand angiogenesis in vivo, by directing the adhesion and extravastationof tumor cells to and from the vasculature to distant tissue siteswithin the body. Moreover, Beg and Baltimore, Science 274:782 (1996),teaches that NF-κB is an anti-apoptotic controlling factor, andinhibition of NF-κB activation makes cells more sensitive toenvironmental stress and cytotoxic agents.

The proteasome inhibitor VELCADE® (bortezomib;N-2-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid) is the firstproteasome inhibitor to achieve regulatory approval. Mitsiades et al.,Current Drug Targets, 7:1341 (2006), reviews the clinical studiesleading to the approval of bortezomib for the treatment of multiplemyeloma patients who have received at least one prior therapy. Fisher etal., J. Clin. Oncol., 30:4867, describes an international multi-centerPhase II study confirming the activity of bortezomib in patients withrelapsed or refractory mantle cell lymphoma. Ishii et al., Anti-CancerAgents in Medicinal Chemistry, 7:359 (2007), and Roccaro et al., Curr.Pharm. Biotech., 7:1341 (2006), discuss a number of molecular mechanismsthat may contribute to the antitumor activities of bortezomib.

As evidenced by the above references, the proteasome represents animportant target for therapeutic intervention. There is thus acontinuing need for new and/or improved proteasome inhibitors.

DESCRIPTION OF THE INVENTION

The present invention provides compounds that are effective inhibitorsof the proteasome. These compounds are useful for inhibiting proteasomeactivity in vitro and in vivo, and are especially useful for thetreatment of various cell proliferative diseases.

Compounds of the invention are of the general formula (I):

or a pharmaceutically acceptable salt or boronic acid anhydride thereof,wherein:

Z¹ and Z² are each independently hydroxy, alkoxy, aryloxy, or aralkoxy;or Z¹ and Z² together form a moiety derived from a boronic acidcomplexing agent; and

Ring A is selected from the group consisting of

Boronic acid compounds of formula (I), wherein Z¹ and Z² are eachhydroxy, are referred to by the following chemical names:

TABLE 1 Proteasome Inhibitors Chemical Name I-1 [(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3- methylbutyl]boronic acid I-2 [(1R)-1-({[(5-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-3 [(1R)-1-({[(3,5-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-4  [(1R)-1-({[(2,5-difluorobenzoyl)amino]acetyl}amino)-3- methylbutyl] boronic acid I-5 [(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-6  [(1R)-1-({[(2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-7  [(1R)-1-({[(2-chloro-5-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-8  [(1R)-1-({[(4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-9   [(1R)-1-({[(3,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronic acid I-10[(1R)-1-({[(3-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-11 [(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronic cid I-12[(1R)-1-({[(3,4-dichlorobenzoyl)amino]acetyl}amino)-3- methylbutyl]boronic acid I-13[(1R)-1-({[(3-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-14 [(1R)-1-({[(2-chloro-4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-15[(1R)-1-({[(2,3-dichlorobenzoyl)amino]acetyl}amino)-3- methylbutyl]boronic acid I-16[(1R)-1-({[(2-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-17 [(1R)-1-({[(2,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronic acid I-18[(1R)-1-({[(4-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid I-19[(1R)-1-({[(4-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronicacid I-20 [(1R)-1-({[(2,4-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl] boronic acid I-21[(1R)-1-({[(3,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronic acid

The term “alkyl”, used alone or as part of a larger moiety, refers to astraight or branched chain or cyclic aliphatic group having from 1 to 12carbon atoms. The term “alkoxy” refers to an —O-alkyl radical.

The terms “aryl” and “ar-”, used alone or as part of a larger moiety,e.g., “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refer to a C₆ to C₁₄aromatic hydrocarbon, comprising one to three rings, each of which isoptionally substituted. Preferably, the aryl group is a C₆₋₁₀ arylgroup. Aryl groups include, without limitation, phenyl, naphthyl, andanthracenyl. An “aralkyl” or “arylalkyl” group comprises an aryl groupcovalently attached to an alkyl group, either of which independently isoptionally substituted. Preferably, the aralkyl group is C₆₋₁₀aryl(C₁₋₆)alkyl, C₆₋₁₀ aryl(C₁₋₄)alkyl, or C₆₋₁₀ aryl(C₁₋₃)alkyl,including, without limitation, benzyl, phenethyl, and naphthylmethyl.

The term “substituted”, as used herein, means that a hydrogen radical ofthe designated moiety is replaced with the radical of a specifiedsubstituent, provided that the substitution results in a stable orchemically feasible compound. Nonlimiting examples of suitablesubstituents include C₁₋₆ alkyl, C₃₋₈ cycloalkyl,C¹⁻⁶alkyl(C₃₋₈)cycloalkyl, C₂₋₈ alkenyl, C₂₋₈ alkynyl, cyano, amino,C₁₋₆ alkylamino, di(C₁₋₆)alkylamino, benzylamino, dibenzylamino, nitro,carboxy, carbo(C₁₋₆)alkoxy, trifluoromethyl, halogen, C₁₋₆ alkoxy, C₆₋₁₀aryl, C₆₋₁₀ aryl(C₁₋₆)alkyl, C₆₋₁₀ aryl(C₁₋₆)alkoxy, hydroxy, C₁₋₆alkylthio, C₁₋₆ alkylsulfinyl, C₁₋₆ alkylsulfonyl, C₆₋₁₀ arylthio, C₆₋₁₀arylsulfinyl, C₆₋₁₀ arylsulfonyl, C₆₋₁₀ aryl, C₁₋₆ alkyl(C₆₋₁₀)aryl, andhalo(C₆₋₁₀)aryl.

The phrase “one or more substituents”, as used herein, refers to anumber of substituents that equals from one to the maximum number ofsubstituents possible based on the number of available bonding sites,provided that the above conditions of stability and chemical feasibilityare met. Unless otherwise indicated, an optionally substituted group mayhave a substituent at each substitutable position of the group, and thesubstituents may be either the same or different. As used herein, theterm “independently selected” means that the same or different valuesmay be selected for multiple instances of a given variable in a singlecompound.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%.

As used herein, the term “comprises” means “includes, but is not limitedto.”

Unless otherwise stated, structures depicted herein are meant to includecompounds which differ only in the presence of one or more isotopicallyenriched atoms. For example, compounds having the present structureexcept for the replacement of a hydrogen atom by a deuterium or tritium,or the replacement of a carbon atom by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of the invention.

As used herein, the term “boronic acid” refers to a chemical compoundcontaining a —B(OH)₂ moiety. In some embodiments, boronic acid compoundscan form oligomeric anhydrides by dehydration of the boronic acidmoiety. For example, Snyder et al., J. Am. Chem. Soc. 80:3611 (1958),reports oligomeric arylboronic acids.

As used herein, the term “boronic acid anhydride” refers to a chemicalcompound formed by combination of two or more molecules of a boronicacid compound, with loss of one or more water molecules. When mixed withwater, the boronic acid anhydride compound is hydrated to release thefree boronic acid compound. In various embodiments, the boronic acidanhydride can comprise two, three, four, or more boronic acid units, andcan have a cyclic or linear configuration. Non-limiting examples ofoligomeric boronic acid anhydrides of peptide boronic acids compound ofthe invention are illustrated below:

In formulae (1) and (2), the variable n is an integer from 0 to about10, preferably 0, 1, 2, 3, or 4. In some embodiments, the boronic acidanhydride compound comprises a cyclic trimer (“boroxine”) of formula(2), wherein n is 1. The variable W has the formula (3):

wherein Ring A has the values described above for formula (I).

In some embodiments, at least 80% of the boronic acid present in theboronic acid anhydride compound exists in a single oligomeric anhydrideform. In some embodiments, at least 85%, 90%, 95%, or 99% of the boronicacid present in the boronic acid anhydride compound exists in a singleoligomeric anhydride form. In certain preferred embodiments, the boronicacid anhydride compound consists of, or consists essentially of, aboroxine having formula (3).

The boronic acid anhydride compound preferably can be prepared from thecorresponding boronic acid by exposure to dehydrating conditions,including, but not limited to, recrystallization, lyophilization,exposure to heat, and/or exposure to a drying agent. Nonlimitingexamples of suitable recrystallization solvents include ethyl acetate,dichloromethane, hexanes, ether, acetonitrile, ethanol, and mixturesthereof.

In some embodiments, Z¹ and Z² together form a moiety derived from aboronic acid complexing agent. For purposes of the invention, the term“boronic acid complexing agent” refers to any compound having at leasttwo functional groups, each of which can form a covalent bond withboron. Nonlimiting examples of suitable functional groups include aminoand hydroxyl. In some embodiments, at least one of the functional groupsis a hydroxyl group. The term “moiety derived from a boronic acidcomplexing agent” refers to a moiety formed by removing the hydrogenatoms from two functional groups of a boronic acid complexing agent.

As used herein, the terms “boronate ester” and “boronic ester” are usedinterchangeably and refer to a chemical compound containing a —B(Z¹)(Z²)moiety, wherein at least one of Z¹ or Z² is alkoxy, aralkoxy, oraryloxy; or Z¹ and Z² together form a moiety derived from a boronic acidcomplexing agent having at least one hydroxyl group.

In some embodiments, Z¹ and Z² together form a moiety derived from acompound having at least two hydroxyl groups separated by at least twoconnecting atoms in a chain or ring, said chain or ring comprisingcarbon atoms and, optionally, a heteroatom or heteroatoms which can beN, S, or O, wherein the atom attached to boron in each case is an oxygenatom.

As employed herein, the term “compound having at least two hydroxylgroups” refers to any compound having two or more hydroxyl groups. Forpurposes of the invention, the two hydroxyl groups preferably areseparated by at least two connecting atoms, preferably from about 2 toabout 5 connecting atoms, more preferably 2 or 3 connecting atoms. Forconvenience, the term “dihydroxy compound” may be used to refer to acompound having at least two hydroxyl groups, as defined above. Thus, asemployed herein, the term “dihydroxy compound” is not intended to belimited to compounds having only two hydroxyl groups. The moiety derivedfrom a compound having at least two hydroxyl groups may be attached toboron by the oxygen atoms of any two of its hydroxyl groups. Preferably,the boron atom, the oxygen atoms attached to boron, and the atomsconnecting the two oxygen atoms together form a 5- or 6-membered ring.

For purposes of the present invention, the boronic acid complexing agentpreferably is pharmaceutically acceptable, i.e., suitable foradministration to humans. In some preferred embodiments, the boronicacid complexing agent is a sugar. The term “sugar” includes anypolyhydroxy carbohydrate moiety, including monosaccharides,disaccharides, polysaccharides, sugar alcohols and amino sugars. In someembodiments, the sugar is a monosaccharide, disaccharide, sugar alcohol,or amino sugar. Non-limiting examples of suitable sugars includeglucose, sucrose, fructose, trehalose, mannitol, sorbitol, glucosamine,and N-methylglucosamine. In certain embodiments, the sugar is mannitolor sorbitol. Thus, in the embodiments wherein the sugar is mannitol orsorbitol, Z¹ and Z² together form a moiety of formula C₆H₁₂O₆, whereinthe oxygen atoms of the two deprotonated hydroxyl groups form covalentattachments with boron to form a boronate ester compound. In certainparticular embodiments, Z¹ and Z² together form a moiety derived fromD-mannitol.

In some embodiments, the compound of formula (I) is formulated as alyophilized powder, as described in Plamondon et al., WO 02/059131,hereby incorporated by reference in its entirety. In some embodiments,the lyophilized powder also comprises free dihydroxy compound.Preferably, the free dihydroxy compound and the compound of formula (I)are present in the mixture in a molar ratio ranging from about 0.5:1 toabout 100:1, more preferably from about 5:1 to about 100:1. In variousembodiments wherein the dihydroxy compound is mannitol, the lyophilizedpowder comprises free mannitol and mannitol boronate ester in a molarratio ranging from about 10:1 to about 100:1, from about 20:1 to about100:1, or from about 40:1 to about 100:1.

In some embodiments, the lyophilized powder comprises mannitol and acompound of formula (I), substantially free of other components.However, the composition can further comprise one or more otherpharmaceutically acceptable excipients, carriers, diluents, fillers,salts, buffers, stabilizers, solubilizers, and other materials wellknown in the art. The preparation of pharmaceutically acceptableformulations containing these materials is described in, e.g.,Remington: The Science and Practice of Pharmacy, 20th Ed., ed. A.Gennaro, Lippincott Williams & Wilkins, 2000, or latest edition.

The lyophilized powder comprising the compound of formula (I) preferablyis prepared according to the procedures described in Plamondon et al.,WO 02/059131. Thus, in some embodiments, the method for preparing thelyophilized powder comprises: (a) preparing an aqueous mixturecomprising a peptide boronic acid and a dihydroxy compound; and (b)lyophilizing the mixture.

General Synthetic Methodology

The compounds of formula (I) can be prepared by methods known to one ofordinary skill in the art. See, e.g., Adams et. al., U.S. Pat. No.5,780,454; Pickersgill et al., International Patent Publication WO2005/097809. An exemplary synthetic route is set forth in Scheme 1below.

Coupling of compound i with an N-protected glycine (ii), followed byN-terminal deprotection, provides compound iii. Examples of suitableprotecting groups (PG) include, without limitation, acyl protectinggroups, e.g., formyl, acetyl (Ac), succinyl (Suc), and methoxysuccinyl;and urethane protecting groups, e.g., tert-butoxycarbonyl (Boc),benzyloxycarbonyl (Cbz), and fluorenylmethoxycarbonyl (Fmoc). Thepeptide coupling reaction can be conducted by prior conversion of thecarboxylic acid moiety of compound ii to an activated ester, e.g., anO—(N-hydroxysuccinnimide) ester, followed by treatment with compound i.Alternatively, the activated ester can be generated in situ bycontacting the carboxylic acid with a peptide coupling reagent. Examplesof suitable peptide coupling reagents include, without limitation,carbodiimide reagents, e.g., dicyclohexylcarbodiimide (DCC) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); phosphoniumreagents, e.g., benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP); and uranium reagents, e.g.,O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramthyluronium tetrafluoroborate(TBTU).

Compound iii is then coupled with a substituted benzoic acid (ArCO₂H) toafford compound iv. The peptide coupling conditions described above forthe coupling of compounds i and ii are also suitable for couplingcompound iii with ArCO₂H. Deprotection of the boronic acid moiety thenaffords compound v. The deprotection step preferably is accomplished bytransesterification in a biphasic mixture comprising the boronic estercompound Iv, an organic boronic acid acceptor, a lower alkanol, a C₅₋₈hydrocarbon solvent, and aqueous mineral acid.

Alternatively, the order of coupling reactions can be reversed, as shownin Scheme 2. Thus, an O-protected glycine (vi) is first coupled with asubstituted benzoic acid (ArCO₂H), followed by ester hydrolysis, to formcompound vii. Coupling with compound i and boronic acid deprotection arethen accomplished as described above for Scheme 1 to afford compound v.

Uses, Formulation, and Administration

The present invention provides compounds that are potent inhibitors ofthe proteasome. The compounds can be assayed in vitro or in vivo fortheir ability to inhibit proteasome-mediated peptide hydrolysis orprotein degradation.

In another aspect, therefore, the invention provides a method forinhibiting one or more peptidase activities of a proteasome in a cell,comprising contacting a cell in which proteasome inhibition is desiredwith a compound described herein, or a pharmaceutically acceptable salt,boronic ester, or boronic acid anhydride thereof.

The invention also provides a method for inhibiting cell proliferation,comprising contacting a cell in which such inhibition is desired with acompound described herein. The phrase “inhibiting cell proliferation” isused to denote the ability of a compound of the invention to inhibitcell number or cell growth in contacted cells as compared to cells notcontacted with the inhibitor. An assessment of cell proliferation can bemade by counting cells using a cell counter or by an assay of cellviability, e.g., an MTT or WST assay. Where the cells are in a solidgrowth (e.g., a solid tumor or organ), such an assessment of cellproliferation can be made by measuring the growth, e.g., with calipers,and comparing the size of the growth of contacted cells withnon-contacted cells.

Preferably, the growth of cells contacted with the inhibitor is retardedby at least about 50% as compared to growth of non-contacted cells. Invarious embodiments, cell proliferation of contacted cells is inhibitedby at least about 75%, at least about 90%, or at least about 95% ascompared to non-contacted cells. In some embodiments, the phrase“inhibiting cell proliferation” includes a reduction in the number ofcontacted cells, as compare to non-contacted cells. Thus, a proteasomeinhibitor that inhibits cell proliferation in a contacted cell mayinduce the contacted cell to undergo growth retardation, to undergogrowth arrest, to undergo programmed cell death (i.e., apoptosis), or toundergo necrotic cell death.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of formula (I), or a pharmaceutically acceptablesalt or boronic acid anhydride thereof, and a pharmaceuticallyacceptable carrier.

If a pharmaceutically acceptable salt of the compound of the inventionis utilized in these compositions, the salt preferably is derived froman inorganic or organic acid or base. For reviews of suitable salts,see, e.g., Berge et al, J. Pharm. Sci. 66:1-19 (1977) and Remington: TheScience and Practice of Pharmacy, 20th Ed., ed. A. Gennaro, LippincottWilliams & Wilkins, 2000.

Nonlimiting examples of suitable acid addition salts include thefollowing: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphor sulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, lucoheptanoate, glycerophosphate, hemisulfate, heptanoate,hexanoate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate and undecanoate.

Suitable base addition salts include, without limitation, ammoniumsalts, alkali metal salts, such as lithium, sodium and potassium salts;alkaline earth metal salts, such as calcium and magnesium salts; othermultivalent metal salts, such as zinc salts; salts with organic bases,such as dicyclohexylamine, N-methyl-D-glucamine, t-butylamine, ethylenediamine, ethanolamine, and choline; and salts with amino acids such asarginine, lysine, and so forth. In some embodiments, thepharmaceutically acceptable salt is a base addition salt of a boronicacid compound of formula (I), wherein Z¹ and Z² are both hydroxy.

The term “pharmaceutically acceptable carrier” is used herein to referto a material that is compatible with a recipient subject, preferably amammal, more preferably a human, and is suitable for delivering anactive agent to the target site without terminating the activity of theagent. The toxicity or adverse effects, if any, associated with thecarrier preferably are commensurate with a reasonable risk/benefit ratiofor the intended use of the active agent.

The terms “carrier”, “adjuvant”, or “vehicle” are used interchangeablyherein, and include any and all solvents, diluents, and other liquidvehicles, dispersion or suspension aids, surface active agents, pHmodifiers, isotonic agents, thickening or emulsifying agents,preservatives, solid binders, lubricants and the like, as suited to theparticular dosage form desired. Remington: The Science and Practice ofPharmacy, 20th Ed., ed. A. Gennaro, Lippincott Williams & Wilkins, 2000discloses various carriers used in formulating pharmaceuticallyacceptable compositions and known techniques for the preparationthereof. Except insofar as any conventional carrier medium isincompatible with the compounds of the invention, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticallyacceptable composition, its use is contemplated to be within the scopeof this invention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins,such as human serum albumin, buffer substances such as phosphates,carbonates, magnesium hydroxide and aluminum hydroxide, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, pyrogen-free water, salts or electrolytessuch as protamine sulfate, disodium hydrogen phosphate, potassiumhydrogen phosphate, sodium chloride, and zinc salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose, sucrose, and mannitol, starches such as corn starchand potato starch, cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate, powderedtragacanth; malt, gelatin, talc, excipients such as cocoa butter andsuppository waxes, oils such as peanut oil, cottonseed oil, saffloweroil, sesame oil, olive oil, corn oil and soybean oil, glycols such aspropylene glycol and polyethylene glycol, esters such as ethyl oleateand ethyl laurate, agar, alginic acid, isotonic saline, Ringer'ssolution, alcohols such as ethanol, isopropyl alcohol, hexadecylalcohol, and glycerol, cyclodextrins such as hydroxypropylβ-cyclodextrin and sulfobutylether β-cyclodextrin, lubricants such assodium lauryl sulfate and magnesium stearate, petroleum hydrocarbonssuch as mineral oil and petrolatum. Coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The pharmaceutical compositions of the invention can be manufactured bymethods well known in the art such as conventional granulating, mixing,dissolving, encapsulating, lyophilizing, or emulsifying processes, amongothers. Compositions may be produced in various forms, includinggranules, precipitates, or particulates, powders, including freezedried, rotary dried or spray dried powders, amorphous powders, tablets,capsules, syrup, suppositories, injections, emulsions, elixirs,suspensions or solutions.

According to a preferred embodiment, the compositions of this inventionare formulated for pharmaceutical administration to a mammal, preferablya human being. Such pharmaceutical compositions of the present inventionmay be administered orally, parenterally, by inhalation spray,topically, rectally, nasally, buccally, vaginally or via an implantedreservoir. The term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,infrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intravenously, or subcutaneously. The formulationsof the invention may be designed to be short-acting, fast-releasing, orlong-acting. Still further, compounds can be administered in a localrather than systemic means, such as administration (e.g., by injection)at a tumor site.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, cyclodextrins, dimethylformamide, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor, and sesameoils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols andfatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Theinjectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, or by incorporating sterilizingagents in the form of sterile solid compositions which can be dissolvedor dispersed in sterile water or other sterile injectable medium priorto use. Compositions formulated for parenteral administration may beinjected by bolus injection or by timed push, or may be administered bycontinuous infusion.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents such as phosphates orcarbonates.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

In some embodiments, the compound of formula (I) is administeredintravenously. In such embodiments, the compound of formula (I) whereinZ¹ and Z² together form a moiety derived from a boronic acid complexingagent can be prepared in the form of a lyophilized powder, as describedabove. The lyophilized powder preferably is reconstituted by adding anaqueous solvent suitable for pharmaceutical administrations. Examples ofsuitable reconstitution solvents include, without limitation, water,saline, and phosphate buffered saline (PBS). Preferably, the lyophilizedpowder is reconstituted with normal (0.9%) saline. Upon reconstitution,an equilibrium is established between a boronate ester compound and thecorresponding free boronic acid compound. In some embodiments,equilibrium is reached quickly, e.g., within 10-15 minutes, after theaddition of aqueous medium. The relative concentrations of boronateester and boronic acid present at equilibrium is dependent uponparameters such as, e.g., the pH of the solution, temperature, thenature of the boronic acid complexing agent, and the ratio of boronicacid complexing agent to boronate ester compound present in thelyophilized powder.

The pharmaceutical compositions of the invention preferably areformulated for administration to a patient having, or at risk ofdeveloping or experiencing a recurrence of, a proteasome-mediateddisorder. The term “patient”, as used herein, means an animal,preferably a mammal, more preferably a human. Preferred pharmaceuticalcompositions of the invention are those formulated for oral,intravenous, or subcutaneous administration. However, any of the abovedosage forms containing a therapeutically effective amount of a compoundof the invention are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention. In someembodiments, the pharmaceutical composition of the invention may furthercomprise another therapeutic agent. In some embodiments, such othertherapeutic agent is one that is normally administered to patients withthe disease or condition being treated.

By “therapeutically effective amount” is meant an amount sufficient tocause a detectable decrease in proteasome activity or the severity of aproteasome-mediated disorder. The amount of proteasome inhibitor neededwill depend on the effectiveness of the inhibitor for the given celltype and the length of time required to treat the disorder. It shouldalso be understood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health, sex, and diet of the patient, time of administration,rate of excretion, drug combinations, the judgment of the treatingphysician, and the severity of the particular disease being treated. Theamount of additional therapeutic agent present in a composition of thisinvention typically will be no more than the amount that would normallybe administered in a composition comprising that therapeutic agent asthe only active agent. Preferably, the amount of additional therapeuticagent will range from about 50% to about 100% of the amount normallypresent in a composition comprising that agent as the onlytherapeutically active agent.

In another aspect, the invention provides a method for treating apatient having, or at risk of developing or experiencing a recurrenceof, a proteasome-mediated disorder. As used herein, the term“proteasome-mediated disorder” includes any disorder, disease orcondition which is caused or characterized by an increase in proteasomeexpression or activity, or which requires proteasome activity. The term“proteasome-mediated disorder” also includes any disorder, disease orcondition in which inhibition of proteasome activity is beneficial.

For example, compounds and pharmaceutical compositions of the inventionare useful in treatment of disorders mediated via proteins (e.g., NFκB,p27^(Kip), p21^(WAF/CIP1), p53) which are regulated by proteasomeactivity. Relevant disorders include inflammatory disorders (e.g.,rheumatoid arthritis, inflammatory bowel disease, asthma, chronicobstructive pulmonary disease (COPD), osteoarthritis, dermatosis (e.g.,atopic dermatitis, psoriasis)), vascular proliferative disorders (e.g.,atherosclerosis, restenosis), proliferative ocular disorders (e.g.,diabetic retinopathy), benign proliferative disorders (e.g.,hemangiomas), autoimmune diseases (e.g., multiple sclerosis, tissue andorgan rejection), as well as inflammation associated with infection(e.g., immune responses), neurodegenerative disorders (e.g., Alzheimer'sdisease, Parkinson's disease, motor neurone disease, neuropathic pain,triplet repeat disorders, astrocytoma, and neurodegeneration as resultof alcoholic liver disease), ischemic injury (e.g., stroke), andcachexia (e.g., accelerated muscle protein breakdown that accompaniesvarious physiological and pathological states, (e.g., nerve injury,fasting, fever, acidosis, HIV infection, cancer affliction, and certainendocrinopathies)).

The compounds and pharmaceutical compositions of the invention areparticularly useful for the treatment of cancer. As used herein, theterm “cancer” refers to a cellular disorder characterized byuncontrolled or disregulated cell proliferation, decreased cellulardifferentiation, inappropriate ability to invade surrounding tissue,and/or ability to establish new growth at ectopic sites. The term“cancer” includes, but is not limited to, solid tumors and bloodbornetumors. The term “cancer” encompasses diseases of skin, tissues, organs,bone, cartilage, blood, and vessels. The term “cancer” furtherencompasses primary and metastatic cancers.

Non-limiting examples of solid tumors that can be treated with thedisclosed proteasome inhibitors include pancreatic cancer; bladdercancer; colorectal cancer; breast cancer, including metastatic breastcancer; prostate cancer, including androgen-dependent andandrogen-independent prostate cancer; renal cancer, including, e.g.,metastatic renal cell carcinoma; hepatocellular cancer; lung cancer,including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolarcarcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer,including, e.g., progressive epithelial or primary peritoneal cancer;cervical cancer; gastric cancer; esophageal cancer; head and neckcancer, including, e.g., squamous cell carcinoma of the head and neck;melanoma; neuroendocrine cancer, including metastatic neuroendocrinetumors; brain tumors, including, e.g., glioma, anaplasticoligodendroglioma, adult glioblastoma multiforme, and adult anaplasticastrocytoma; bone cancer; and soft tissue sarcoma.

Non-limiting examples of hematologic malignancies that can be treatedwith the disclosed proteasome inhibitors include acute myeloid leukemia(AML); chronic myelogenous leukemia (CML), including accelerated CML andCML blast phase (CML-BP); acute lymphoblastic leukemia (ALL); chroniclymphocytic leukemia (CLL); Hodgkin's disease (HD); non-Hodgkin'slymphoma (NHL), including follicular lymphoma and mantle cell lymphoma;B-cell lymphoma; T-cell lymphoma; multiple myeloma (MM); Waldenstrom'smacroglobulinemia; myelodysplastic syndromes (MDS), including refractoryanemia (RA), refractory anemia with ringed siderblasts (RARS),(refractory anemia with excess blasts (RAEB), and RAEB in transformation(RAEB-T); and myeloproliferative syndromes.

In some embodiments, the compound or composition of the invention isused to treat a patient having or at risk of developing or experiencinga recurrence in a cancer selected from the group consisting of multiplemyeloma and mantle cell lymphoma.

In some embodiments, the proteasome inhibitor of the invention isadministered in conjunction with another therapeutic agent. The othertherapeutic agent may also inhibit the proteasome, or may operate by adifferent mechanism. In some embodiments, the other therapeutic agent isone that is normally administered to patients with the disease orcondition being treated. The proteasome inhibitor of the invention maybe administered with the other therapeutic agent in a single dosage formor as a separate dosage form. When administered as a separate dosageform, the other therapeutic agent may be administered prior to, at thesame time as, or following administration of the proteasome inhibitor ofthe invention.

In some embodiments, a proteasome inhibitor of formula (I) isadministered in conjunction with an anticancer agent. As used herein,the term “anticancer agent” refers to any agent that is administered toa subject with cancer for purposes of treating the cancer.

Non-limiting examples of DNA damaging chemotherapeutic agents includetopoisomerase I inhibitors (e.g., irinotecan, topotecan, camptothecinand analogs or metabolites thereof, and doxorubicin); topoisomerase IIinhibitors (e.g., etoposide, teniposide, and daunorubicin); alkylatingagents (e.g., melphalan, chlorambucil, busulfan, thiotepa, ifosfamide,carmustine, lomustine, semustine, streptozocin, decarbazine,methotrexate, mitomycin C, and cyclophosphamide); DNA intercalators(e.g., cisplatin, oxaliplatin, and carboplatin); DNA intercalators andfree radical generators such as bleomycin; and nucleoside mimetics(e.g., 5-fluorouracil, capecitibine, gemcitabine, fludarabine,cytarabine, mercaptopurine, thioguanine, pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examplesillustrate how to make or test specific compounds, and are not to beconstrued as limiting the scope of the invention in any way.

EXAMPLES Abbreviations

-   DCM methylene chloride-   DIEA diisopropylethyl amine-   EDCI N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride-   EtOAc ethyl acetate-   h hours-   HPLC high performance liquid chromatography-   TBTU o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium    tetrafluoroborate-   HOBt 1-hydroxybenztriazole hydrate-   LCMS liquid chromatography mass spectrum-   min minutes-   tr retention time from diode array spectra

Analytical LC-MS Methods

Spectra were run on a Symmetry C18-3.5 μm-4.6×50 mm column using thefollowing gradient:

Solvent A: 2% isopropyl alcohol, 98% water, 10 mM NH4OAc

Solvent B: 75% acetonitrile, 25% methanol, 10 mM NH4OAc

Time [min] Flow rate [mL/min] % of solvent B 0.0 1.0 5.0 3.5 1.0 100.04.9 1.0 100.0 5.0 1.0 5.0

Example 1 Synthesis of[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-1)

Step 1: methyl [(2,3-difluorobenzoyl)amino]acetate

To a solution of 2,3-difluorobenzoic acid (0.190 g, 1.2 mmol) intetrahydrofuran (5 mL) were added glycine methyl ester hydrochloride(0.150 g, 1.2 mmol), HOBt (0.162 g, 1.2 mmol), DIEA (0.209 mL, 1.2 mmol)and EDCI (0.252 g, 1.3 mmol). The reaction mixture was allowed to stirovernight. The reaction mixture was quenched with a saturated solutionof sodium bicarbonate and the product partitioned into DCM. Separationof the organic layer followed by removal of the solvent gave methyl[(2,3-difluorobenzoyl)amino]acetate which was used in the next stepwithout purification.

Step 2: [(2,3-difluorobenzoyl)amino]acetic acid

To a solution of methyl [(2,3-difluorobenzoyl)amino]acetate (0.250 g,1.1 mmol) in methanol (7 mL) were added lithium hydroxide (0.053 g, 2.2mmol) and water (3 mL). The reaction mixture was allowed to stirovernight. The mixture was diluted with water (20 mL) and acidified with1N HCl (5 mL). The product was partitioned into DCM/methanol (4:1). Theorganic layer was dried over sodium sulfate and the solvent removed togive [(2,3-difluorobenzoyl)amino]acetic acid which was used in the nextstep without purification.

Step 3:2,3-difluoro-N-[2-({(1R)-3-methyl-1-[(3aR,4R,6R,7aS)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide

To a solution of [(2,3-difluorobenzoyl)amino]acetic acid (0.205 g, 0.95mmol) in dimethylformamide (10 mL) were added TBTU (0.337 g, 1.0 mmol)and(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butan-1-amineas its trifluoroacetate salt (0.362 g, 0.95 mmol). The mixture wasallowed to cool to 0° C. and DIEA (0.498 mL, 2.9 mmol) was addeddropwise. The reaction mixture was allowed to warm to room temperatureand stirred overnight. The reaction was quenched with water (100 mL) andthe product partitioned into DCM. The organic layer was dried oversodium sulfate and the solvent removed to give2,3-difluoro-N-[2-({(1R)-3-methyl-1-[(3aR,4R,6R,7aS)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide.

Step 4:[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid

To a solution of2,3-difluoro-N-[2-({(1R)-3-methyl-1-[(3aR,4R,6R,7aS)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide(0.536 g, 1.2 mmol) in methanol/1N HCl (1:1) (1.5 mL) were addedheptanol (1 mL) and isobutyl boronate (0.207 g, 2.0 mmol). The reactionmixture was allowed to stir overnight. The heptanol layer was separatedand the methanol/HCl layer was concentrated. The crude product waspurified by reverse phase HPLC to give[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid.

Step 5:[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-1)

To a solution of[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid (0.085 g, 0.26 mmol) in t-butyl alcohol (2 mL) and water (5 mL) wasadded D-mannitol (0.943 g, 5.2 mmol). The solution was warmed andallowed to stir until everything dissolved. The solution was then frozenand the solvent removed by lyophilization to give[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-1) (0.98 g, 97%).

Example 2 Synthesis of[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-5)

Step 1: tert-butyl[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]carbamate

To a mixture of(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butan-1-amineas its trifluoroacetate salt (4.9 g, 10.8 mmol),N-α-(tert-Butoxycarbonyl)glycine (1.98 g, 11.3 mmol) and TBTU (3.81 g,11.9 mmol) in DCM (100 mL) was added dropwise over 15 min a solution ofDIEA (5.64 mL, 32.4 mmol) in DCM (25 mL). The reaction mixture wasallowed to stir overnight and was concentrated. The crude product waspurified by column chromatography to give tert-butyl[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]carbamate(2.5 g, 55%).

Step 2:2-amino-N-{(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}acetamide

To a solution of tert-butyl[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]carbamate(2.5 g, 5.9 mmol) in DCM (15 mL) was added 4M HCl in dioxane (5.9 mL).The reaction mixture was allowed to stir for 2 h and concentrated togive2-amino-N-{(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}acetamidewhich was used in the next step without purification.

Step 3:2-bromo-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide

To a solution of 2-bromobenzoic acid (0.124 g, 0.62 mmol) in DCM (2.25mL) were added EDCI (0.119 g, 0.62 mmol), HOBt (0.084 g, 0.62 mmol),N-methyl morpholine (0.185 mL, 1.68 mmol) and2-amino-N-{(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}acetamide(0.2 g, 0.56 mmol). The reaction mixture was allowed to stir for 2 h andwas concentrated. The residue was diluted with water and extracted withEtOAc. The organic solutions were combined, washed with brine, driedover MgSO₄, filtered and concentrated. The crude product was purified bycolumn chromatography to give2-bromo-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide(0.22 g, 78%).

Step 4:[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid

To a solution of2-bromo-N-[2-({(1R)-3-methyl-1-[(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methano-1,3,2-benzodioxaborol-2-yl]butyl}amino)-2-oxoethyl]benzamide(0.220 g, 0.44 mmol) in methanol/hexane (1:1) (2.2 mL) were added 1N HCl(1 mL, 1.0 mmol) and isobutyl boronate (0.078 g, 0.76 mmol). Thereaction mixture was allowed to stir overnight. The reaction mixture wasconcentrated and purified by reverse phase HPLC to give[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid (0.119 g, 73%).

Step 5:[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-5)

To a solution of[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid (0.103 g, 0.28 mmol) in tert-butyl alcohol (9 mL) and water (15 mL)was added D-mannitol (1.01 g, 5.5 mmol). The solution was warmed andallowed to stir until everything dissolved. The solution was then frozenand the solvent removed by lyophilization to give[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid. 20 D-mannitol (I-5) (0.92 g, 84%).

Compounds in the following table were prepared from the appropriatestarting materials in a method analogous to that of Example 1 or 2:

I-1  LCMS: ES- 327.3, tr = 3.36 min. I-2  LCMS: ES- 343.2, tr = 3.62min. I-3  LCMS: ES- 327.3, tr = 3.49 min. I-4  LCMS: ES- 327.3, tr =3.27 min. I-5  LCMS: ES- 369.2, tr = 3.30 min. ¹H NMR (300 MHz, d₄-MeOD)δ: 7.62 (dd, 1H), 7.28-7.50 (m, 3H), 4.19 (s, 2H), 2.70-2.78 (m, 1H),1.57-1.71 (m, 1H), 1.26- 1.40 (m, 2H) and 0.89 (d, 6H). I-6  LCMS: ES-309.1, tr = 3.14 min. I-7  LCMS: ES- 343.2, tr = 3.30 min. I-8  LCMS:ES- 309.3, tr = 3.23 min. I-9  LCMS: ES- 327.3, tr = 3.49 min. I-10LCMS: ES- 325.2, tr = 3.58 min. I-11 LCMS: ES- 359.2, tr = 3.66 min. ¹HNMR (300 MHz, d₄-MeOD) δ: 7.62 (s, 1H), 7.49 (d, 2H), 4.23 (s, 2H),2.74-2.82 (m, 1H), 1.62-1.78 (m, 1H), 1.30-1.45 (m, 2H) and 0.95 (d,6H). I-12 LCMS: ES- 359.2, tr = 3.95 mm. I-13 LCMS: ES- 309.2, tr = 3.34min. I-14 LCMS: ES- 343.2, tr = 3.44 min. I-15 LCMS: ES- 359.2, tr =3.26 min. I-16 LCMS: ES- 325.2, tr = 3.20 min. I-17 LCMS: ES- 327.3, tr= 3.39 min. I-18 LCMS: ES- 343.2, tr = 3.58 min. I-19 LCMS: ES- 325.1,tr = 3.51 min. I-20 LCMS: ES- 359.2, tr = 3.54 min. I-21 LCMS: ES-359.2, tr = 3.99 min.

Example 2 20S Proteasome Assay

To 1 μL of test compound dissolved in DMSO in a 384-well blackmicrotiter plate is added 25 μL of assay buffer at 37° C. containinghuman PA28 activator (Boston Biochem, 12 nM final) with Ac-WLA-AMC (β5selective substrate) (15 μM final), followed by 25 μL of assay buffer at37° C. containing human 20S proteasome (Boston Biochem, 0.25 nM final).Assay buffer is composed of 20 mM HEPES, 0.5 mM EDTA and 0.01% BSA,pH7.4. The reaction is followed on a BMG Galaxy plate reader (37° C.,excitation 380 nm, emission 460 nm, gain 20). Percent inhibition iscalculated relative to 0% inhibition (DMSO) and 100% inhibition (10 μMbortezomib) controls.

When tested in this assay, compounds I-1 to I-21 all exhibited IC₅₀values less than 50 nM.

Example 3 Antiproliferation Assay

HCT-116 (1000) or other tumor cells in 100 μL of appropriate cellculture medium (McCoy's 5A for HCT-116, Invitrogen) supplemented with10% fetal bovine serum (Invitrogen) are seeded in wells of a 96-wellcell culture plate and incubated overnight at 37° C. Test compounds areadded to the wells and the plates are incubated for 96 hours at 37° C.MIT or WST reagent (10 μL, Roche) are added to each well and incubatedfor 4 hours at 37° C. as described by the manufacturer. For MIT themetabolized dye is solubilized overnight according to manufacturer'sinstructions (Roche). The optical density for each well is read at 595nm (primary) and 690 nm (reference) for the MIT and 450 nm for the WSTusing a spectrophotometer (Molecular Devices). For the MTT the referenceoptical density values are subtracted from the values of the primarywavelength. Percent inhibition is calculated using the values from aDMSO control set to 100%.

Example 4 In Vivo Tumor Efficacy Model

Freshly dissociated HCT-116 (2-5×10⁶) or other tumor cells in 100 μL ofRPMI-1640 media (Sigma-Aldrich) are aseptically injected into thesubcutaneous space in the right dorsal flank of female CD-1 nude mice(age 5-8 weeks, Charles River) using a 1 mL 26 3/8-ga needle (BectonDickinson Ref#309625). Alternatively, some xenograft models require theserial passaging of tumor fragments. In these cases, small fragments oftumor tissue (approximately 1 mm³) are implanted subcutaneously in theright dorsal flank of anesthetized (3-5% isoflourane/oxygen mixture)C.B-17/SCID mice (age 5-8 weeks, Charles River) via a 13-ga trocar(Popper & Sons 7927). Beginning at day 7 after inoculation tumors aremeasured twice weekly using a vernier caliper. Tumor volumes arecalculated using standard procedures (0.5×(length×width²)). When thetumors reach a volume of approximately 200 mm³ mice are randomized intotreatment groups and begin receiving drug treatment. Dosing andschedules are determined for each experiment based on previous resultsobtained from pharmacokinetic/pharmacodynamic and maximum tolerated dosestudies. The control group will receive vehicle without any drug.Typically, test compound (100-200 μL) is administered via intravenous(27-ga needle), oral (20-ga gavage needle) or subcutaneous (27-ganeedle) routes at various doses and schedules. Tumor size and bodyweight are measured twice a week and the study is terminated when thecontrol tumors reach approximately 2000 mm³.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, these particular embodiments areto be considered as illustrative and not restrictive. It will beappreciated by one skilled in the art from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention, which is to be defined by theappended claims rather than by the specific embodiments.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. The issued patents, applications,and references that are cited herein are hereby incorporated byreference to the same extent as if each was specifically andindividually indicated to be incorporated by reference. In the case ofinconsistencies, the present disclosure, including definitions, willcontrol.

What is claimed is:
 1. A method for inhibiting proteasome activitycomprising administrating to a patient in need of such inhibition acompound of formula (I), or a pharmaceutical composition comprising acompound of formula (I)

or a pharmaceutically acceptable salt or a boronic acid anhydridethereof, wherein Ring A is selected from the group consisting of

and Z¹ and Z² are each hydroxy, alkoxy, aryloxy, or aralkoxy, or Z¹ andZ² together form a moiety derived from a boronic acid complexing agent.2. The method of claim 1, wherein Z¹ and Z² are each hydroxy.
 3. Themethod of claim 1, wherein Z¹ and Z² together form a moiety derived froma boronic acid complexing agent.
 4. The method of claim 1, wherein RingA is

and Z¹ and Z² are each hydroxy.
 5. The method of claim 1, wherein Ring Ais

and Z¹ and Z² together form a moiety derived from a boronic acidcomplexing agent.
 6. The method of claim 1, wherein the compound offormula (I) is selected from:[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(5-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,5-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,5-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chloro-5-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,4-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chloro-4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,3-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,4-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid; and[(1R)-1-({[(3,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid.
 7. The method of claim 1, wherein the compound is selected from[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid.
 8. The method of claim 1, wherein the patient in need of theinhibition is a patient suffering from cancer.
 9. The method of claim 8,wherein the cancer is multiple myeloma.
 10. The method of claim 8,wherein the cancer is lymphoma.
 11. The method of claim 1, wherein thepatient in need of the inhibition is a patient having or at risk ofdeveloping or experiencing a recurrence in a cancer selected frommultiple myeloma, or lymphoma.
 12. The method of claim 1, wherein thecompound or the pharmaceutical composition is administered with anothertherapeutic agent.
 13. The method of claim 11, wherein the othertherapeutic agent is melphalan.
 14. The method of claim 11, wherein theother therapeutic agent is lenalidomide.
 15. The method of claim 11,wherein the other therapeutic agent is cyclophosphamide.
 16. The methodof claim 1, wherein the compound or the pharmaceutical composition isadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally, or via an implanted reservoir.17. The method of claim 1, wherein the compound or the pharmaceuticalcomposition is administered orally.
 18. The use of claim 1, wherein thecompound or pharmaceutical composition is administered intravenously.19. The method of claim 1, wherein the compound or pharmaceuticalcomposition is administered systemically, or locally.
 20. A method forinhibiting proteasome activity comprising contacting a living cell inwhich such inhibition is desired with a compound of formula (I), or apharmaceutical composition comprising a compound of formula (I)
 21.

or a pharmaceutically acceptable salt or a boronic acid anhydridethereof, wherein Ring A is selected from the group consisting of

and Z¹ and Z² are each hydroxy, alkoxy, aryloxy, or aralkoxy, or Z¹ andZ² together form a moiety derived from a boronic acid complexing agent.22. The method of claim 20, wherein Z¹ and Z² are each hydroxy.
 23. Themethod of claim 20, wherein Z¹ and Z² together form a moiety derivedfrom a boronic acid complexing agent.
 24. The method of claim 20,wherein Ring A is

and Z¹ and Z² are each hydroxy.
 25. The method of claim 20, wherein RingA is

and Z¹ and Z² together form a moiety derived from a boronic acidcomplexing agent.
 26. The method of claim 20, wherein the compound offormula (I) is selected from:[(1R)-1-({[(2,3-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(5-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,5-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,5-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-bromobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chloro-5-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3,4-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(3-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chloro-4-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,3-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,4-difluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-chloro-2-fluorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(4-chlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid;[(1R)-1-({[(2,4-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid; and[(1R)-1-({[(3,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid.
 27. The method of claim 20, wherein the compound is selected from[(1R)-1-({[(2,5-dichlorobenzoyl)amino]acetyl}amino)-3-methylbutyl]boronicacid.